This proposal examines the role of RNase-L as a novel mediator of senescence in vitro, and as a biochemical mechanism of aging in vivo. RNase-L is the ribonuclease component of the interferon-regulated 2-5A pathway. RNase-L functions in antiviral and growth inhibitory activities through the degradation of viral, ribosomal, and messenger RNAs. Constitutive expression of transfected RNase-L results in a decreased mitotic index and senescent morphology. Moreover, the human RNASE- L gene maps to chromosome 1q25, a region thought to harbor gene(s) required for replicative senescence. Based on these finding, we hypothesize that: i) activation of RNASE-L is critical for induction of senescence in vitro, and functions in aging in vivo; ii) RNASE-L promotes the onset of senescence through the post-transcriptional down- regulation of proliferation stimulatory genes and iii) RNASE-L suppresses gene expression through the selective degradation of mRNAs and/or the inhibition of their translation via rRNA cleavage. To determine the role of RNase-L in senescence, we will measure the induction of senescence in human and embryonic mouse fibroblasts in which RNASE-L activity is increased or inhibited (aim 1). The expression and activity of 2-5A pathway enzymes varies in young and aged human fibroblasts and in tissues from young and aged mice (aim 2). This comprehensive survey will incorporate a novel PCR based rRNA cleavage assay to detect RNASE-L activity in intact cells. RNASE-L is predicted to function in senescence via the post-transcriptional modulation of gene expression. We will examine the expression of known senescence regulated genes in cells with reduced or increased RNASE-L activity (aim 3). Genes that exhibit RNASE-L dependent regulation represent candidate substrates and the half-lives of their mRNAs will be determined. This work will increase our understanding of senescent cells by determining: i) the role of RNASE-L as a mediator of senescence; ii) the expression and activity of the 2-5A pathway in aging cells; and iii) how RNASE-L modulates mRNA stability, and thus gene expression, in senescent cells. A comprehensive understanding of cellular senescence is essential to determine its relationship to aging and cancer; such information may lead to the novel therapies.